This invention relates to water-insoluble derivatives of biologically-active enzymes.
The utility of enzymes as catalysts is well established.
Immobilized derivatives of active enzymes are useful as readily removable catalysts for various biochemical reactions such as proteolysis, curdling of milk, conversion of starch to glucose, conversion of glucose to fructose, hydrolysis of pectins, and the like.
It is known to prepare such immobilized protein derivatives by attaching a biologically-active protein such as an enzyme to a carrier which is insoluble in aqueous solutions or which becomes insoluble as a result of the protein attachment. The attachment is made through a functional group of the protein which is not essential for biological activity. In such a manner a biologically-active yet water-insoluble conjugate can be produced. It is known to produce such conjugates by several distinct methods such as adsorption, ion-exchange, formation of a covalent bond between the protein and the carrier, crosslinking, inclusion, and polymerization.
The present invention falls into the general category where a covalent bond is formed between the enzyme and the water-insoluble carrier. Enzymes insolubilized on an inert matrix by covalent bonding can be used as a reactor core for efficient continuous conversion processes of various substrates. By passing a suitable substrate solution through such an enzyme reactor large quantities of the substrate can be exposed to a fixed quantity of the enzyme and thus converted into desired products. The enzymatically active reactor core can be used for long time periods, and there exists no need for a subsequent separation of the enzyme catalyst from the substrate because the enzyme is retained within the reactor and the conversion reaction is automatically stopped when the substrate solution, together with the reaction products, exits from the reactor.
In preparing immobilized enzyme conjugates it is important that the methods utilized to effect covalent bonding of the enzyme to the carrier or matrix do not inactivate the enzyme and do not destroy the structural integrity of the matrix. Enzymes are usually quite unstable and cannot be subjected to severe reaction conditions without adversely affecting activity. As to the carrier or matrix, in a flow-through reactor the liquid permeability of the reactor core is very important from a practical process standpoint and any damage to or weakening of the intended matrix should be avoided.
While it is known to insolubilize proteins such as enzymes by covalent bonding to an inert matrix or carrier, the preparation of a matrix which is reactive toward the enzyme yet suitable for use as a flow-through reactor core is often complicated, laborious, and expensive.
Goldstein et al., Biochemistry 9, No. 11, 2322-2333 (1970), disclose a matrix which is dialdehyde starch crosslinked with a bifunctional reactant such as methylene dianiline in order to render the matrix water-insoluble and in order to provide, by means of bifunctional reagent molecules attached to the crosslinked starch at one point only, reactive sites for coupling to an enzyme. The enzyme is not attached directly to the dialdehyde starch but to the free functional group of the bifunctional reactant by diazotization. While a good, enzymatically-active product, representing an improvement over enzymes attached by diazotization to p-aminobenzyl cellulose (Goldstein et al., p. 2331) can be produced in such a manner, it is readily apparent to one skilled in the art that the preparation of such a product is still involved and expensive.
It is an object of the present invention to provide a biologically-active, water-insoluble dialdehyde celluloseenzyme conjugate having relatively high biological activity yet which can be readily and inexpensively produced.
It is a further object to provide a high-activity flowthrough reactor having a water-insoluble dialdehyde cellulose core with a biologically-active enzyme covalently bonded directly to the core.
It is another object of this invention to provide a method for producing a biologically-active, flow-through reactor.
It is a more specific object of this invention to provide a method for making a liquid permeable cellulose/enzyme conjugate in place, ready for use for treating various liquids which can be passed through the reactor without the necessity for further handling of the cellulose/enzyme product to ready it for use.
It is an additional object to provide a method for firmly and directly bonding a biologically-active enzyme to dialdehyde cellulose.
Yet another object of this invention is to provide a method for covalently bonding an active enzyme, to dialdehyde cellulose without subjecting the protein to severe reaction conditions.
Still other objects of this invention will readily present themselves to the skilled artisan upon reference to the ensuing specification and the claims.